Dengue (DEN) viruses belong to the genus flavivirus, within the family Flaviviridae. There are at least 70 flavivirus species, among which the most important human pathogens are the DEN viruses, yellow fever virus, and the Japanese (JE) and tick-borne encephalitis viruses. Diseases caused by the 4 serotypes of DEN virus (DEN1-4), dengue fever (DF) or dengue hemorrhagic fever/shock syndrome (DHF/DSS), are endemic or epidemic in tropical and sub-tropical countries around the world.
In a manner similar to that of yellow fever, dengue is transmitted between humans by the domestic mosquito vector, Aedes aegypti. Evidence has emerged for the existence of a sylvan cycle of transmission analogous to that of yellow fever, involving monkeys and several sylvatic Aedes species. Transovarial transmission has been demonstrated experimentally in these forest mosquito species.
In light of this evidence, a model of dengue fever transmission where the virus emerges from the jungle to infect urban populations may be suggested. In this model, monkeys serve as a reservoir for the virus and transmission of the virus from monkey to monkey occurs through a mosquito vector. According to this model, the virus is transmitted from mosquito to human and then from that infected human to other mosquitoes of the species Aedes aegypti. Once these mosquitoes are infected, the dengue virus is transmitted to other human hosts. These infected individuals then pass the dengue virus on to other Aedes aegypti mosquitoes and expand the range of infection. Person-to-person spread of dengue virus infection and disease does not occur.
At present, because of several demanding technical problems, there is no vaccine available to prevent the diseases caused by dengue virus infection (DF and DHF/DSS). For example, a suitable live virus vaccine ought not to replicate efficiently in the mosquito vector, since it is conceivable that a live, attenuated vaccine could revert to full virulence during replication in a mosquito that has fed on a vaccinee. Thus, a local or regional immunization program could lead to the spread of illness rather than the diminution of disease incidence in the vaccinated population. Accordingly, a safe and effective dengue virus vaccine will have a severely limited ability to be transmitted from human host to mosquito vector. The present invention seeks to provide such a vaccine and solve the long felt need for safe and effective vaccines directed to the known serotypes of dengue as well as to provide vaccines for other flaviviruses.
The flavivirus genome is a positive-stranded xcx9c11-kb RNA including 5xe2x80x2- and 3xe2x80x2-noncoding regions (NCR) of approximately 100 and 400 to 600 nucleotides, respectively. The 3xe2x80x2-NCR contains adjacent, thermodynamically stable, conserved short and long stem and loop structures (the 3xe2x80x2-SL), formed by the 3xe2x80x2-terminal xcx9c100 nucleotides. The nucleotide sequences within the 3xe2x80x2-SL are not well conserved among species. The requirement for the 3xe2x80x2-SL for replication was examined in the context of dengue virus, type 2, (DEN2) replication, by mutagenesis of an infectious cDNA copy of a DEN2 genome. Genomic full-length RNA was transcribed in vitro and used to transfect monkey kidney cells. A substitution mutation, in which the 3xe2x80x2-terminal 93 nucleotides comprising the wild type DEN2 3xe2x80x2-SL sequence were replaced by the 96 nucleotide sequence of the West Nile virus (WN) 3xe2x80x2-SL, was sublethal for virus replication. Analysis of the growth phenotypes of additional mutant viruses derived from RNAs containing DEN2-WN chimeric 3xe2x80x2-SL structures suggested that the wild type DEN2 nucleotide sequence forming the bottom half of the long stem and loop in the 3xe2x80x2-SL was required for viability. One 7 base pair substitution mutation in this domain resulted in a mutant virus that grew well in monkey kidney cells but was severely restricted in cultured mosquito cells. In contrast, transpositions and/or substitutions of the wild type DEN2 nucleotide sequence in the top half of the long stem and in the short stem and loop were relatively well tolerated, provided the stem-loop secondary structure was conserved. A mutant dengue virus that was observed to be replication-defective in mosquito cells in tissue culture was also shown to be replication-defective in adult mosquitoes. The methods discussed herein also contemplate utility for use against any flavivirus, since the 3xe2x80x2-SL structure is conserved among all the more than 20 flavivirus genomes that have been subjected to nucleotide sequence analysis to date.